Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation, in innate cell clearance of microbial pathogens, and in neural cell maintenance. However, the role of autophagy in T lymphocyte development and survival is not known. Here, we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5−/− chimeric mice, we found that Atg5-deficient T lymphocytes underwent full maturation. However, the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery, Atg5−/− CD8+ T lymphocytes displayed dramatically increased cell death. Furthermore, Atg5−/− CD4+ and CD8+ T cells failed to undergo efficient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation.

Macroautophagy (hereafter referred to as autophagy) is a well-conserved intracellular degradation process. Recent studies examining cells lacking the autophagy genes Atg5 and Atg7 have demonstrated that autophagy plays essential roles in cell survival during starvation, in innate cell clearance of microbial pathogens, and in neural cell maintenance. However, the role of autophagy in T lymphocyte development and survival is not known. Here, we demonstrate that autophagosomes form in primary mouse T lymphocytes. By generating Atg5 −/− chimeric mice, we found that Atg5-defi cient T lymphocytes underwent full maturation. However, the numbers of total thymocytes and peripheral T and B lymphocytes were reduced in Atg5 chimeras. In the periphery, Atg5 −/− CD8 + T lymphocytes displayed dramatically increased cell death. Furthermore, Atg5 −/− CD4 + and CD8 + T cells failed to undergo effi cient proliferation after TCR stimulation. These results demonstrate a critical role for Atg5 in multiple aspects of lymphocyte development and function and suggest that autophagy may be essential for both T lymphocyte survival and proliferation.

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T lymphocyte development and function are tightly regulated by the intrinsic death pathway through members of the Bcl-2 family. Genetic studies have demonstrated that the Bcl-2 family member Mcl-1 is an important anti-apoptotic protein in the development of multiple cell types including T lymphocytes. However, the expression pattern and anti-apoptotic roles of Mcl-1 in T lymphocytes at different developmental stages remain to be fully determined. In this study, we examined the expression pattern of Mcl-1 in different populations of T cells at the single-cell level and found that Mcl-1 protein is constitutively expressed in all T cell populations and up-regulated upon TCR stimulation. We then investigated the role of Mcl-1 in the survival of these different populations by conditionally deleting Mcl-1 at various T cell stages. Our results show that Mcl-1 is required for the survival of double-negative and single-positive thymocytes as well as naive and activated T cells. Furthermore, we demonstrate that Mcl-1 functions together with Bcl-xL to promote double-positive thymocyte survival. Thus, Mcl-1 is a critical anti-apoptotic factor for the survival of T cells at multiple stages in vivo.

Developing thymocytes are screened for self-reactivity before exiting the thymus, but how thymocytes scan the medulla for self-antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 microns in diameter, and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also exhibited confined, slower migration, and may correspond to auto-reactive thymocytes. Our data suggest that many auto-reactive thymocytes do not undergo immediate arrest and death upon encounter with a negative selecting ligand, but rather adopt an altered migration program while remaining within the medullary microenvironment.

Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis: the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work together to regulate T lymphocyte development and function.

Two-photon microscopy and flow cytometry reveal the timing of thymocyte death and the surprisingly close coupling between cell death and phagocytosis during negative selection in thymic slices.

The orphan nuclear receptor, retinoid acid-related orphan receptor (ROR)α, is essential for the development of cerebellar Purkinje cells and bone tissue. RORα may also play a critical role in lymphocyte development and function because staggerer mice, a natural mutant strain with a disrupted expression of RORα, have reduced thymic and splenic cellularity. In this report, we analyzed the role of RORα in lymphocyte development by examining lymphoid compartments in RORα−/− mice and Rag-2−/− mice reconstituted with RORα−/− bone marrow. We found that T and B cell development was severely defective in RORα−/− mice, but not in Rag-2−/−/RORα−/− chimeric mice. We also analyzed cellular and humoral immune responses in Rag-2−/−/RORα−/− chimeric mice. Our results show that serum IgG levels were elevated in Rag-2−/−/RORα−/− chimeric mice after immunization with a T-dependent Ag compared with control chimeras. IFN-γ production by RORα−/− CD8+ T cells after TCR stimulation was also increased. Furthermore, RORα−/− mast cells and macrophages produced an increased amount of TNF-α and IL-6 upon activation. These results indicate that RORα indirectly regulates lymphocyte development by providing an appropriate microenvironment and controls immune responses by negatively regulating cytokine production in innate immune cells and lymphocytes.